Evidence from numerous studies has accumulated that dopamine receptors like other G protein coupled receptors (GPCRs), function within dynamic macromolecular assemblies as oligomers. Conventional thinking about GPCRs existing solely as monomers has been challenged since receptors have been shown to occur as oligomers in cells and in native tissues. More intriguingly, we have discovered that direct association between the D1 and D2 dopamine receptors generates heterooligomeric complexes with novel signaling properties, distinct from the signaling of the constituent receptors. D1-D2 heteromer activation connects dopamine to Gq-linked rapid calcium signaling in brain. We have identified a novel compound that selectively activates the D1-D2 heteromer, without any effect on D1 or D2 homooligomers. The long-term objectives are to elucidate the physiological functions of the D1-D2 receptor complex we identified, within the framework of dopamine biology and to understand its role in drug addiction. We hypothesize that (i) dopamine receptors form heterooligomers to generate unique signaling complexes with novel pharmacology, trafficking and internalization properties, (ii) critical sites for the D1-D2 interaction reside in the D1 receptor carboxyl tail, (iii) the D1-D2 receptor heteromer is a critical mediator of reward in dopamine post-synaptic signaling targeted to the mesolimbic dopamine system. The specific aims are to evaluate the dopamine D1-D2 receptor heterooligomer signaling cascade in cultured neurons using a cameleon calcium sensor, to localize it to the post-synaptic density and to evaluate its effect on CaMKII, BDNF and DARPP-32 expression. Activation of the D1-D2 heteromer selectively in brain will be used to determine its functional correlates biochemically and behaviorally. The physiological interactions between the D1 and D2 receptors in neurons and in brain will be analyzed in situ in brain sections by a novel confocal FRET method, which examines the efficiency of energy transfer and the proximity of D1 and D2 receptors in native tissue. The regulation of the heteromer activated calcium signaling pathway by desensitization, internalization and resensitization in response to dopamine, a selective agonist and drugs of abuse, such as amphetamine and cocaine will be evaluated. Attempts will be made to resolve the structural determinants of the heterooligomeric interaction and to disrupt the heteromer using various strategies in neurons and ultimately in vivo to monitor functional effects behaviorally. The D1- D2 dopamine receptor-signaling complex represents an exciting novel dimension of dopamine function that is as yet unexplored. It will be critical to study, to understand physiological processes related to function of the heteromer, and to eventually incorporate aspects of this knowledge into the search for new therapeutic agents for drug addiction. This research is highly relevant to the mission of NIDA. PUBLIC HEALTH RELEVANCE: This proposed research has relevance for understanding the function of dopamine within the nucleus accumbens, which is an important brain structure involved in the neural circuitry of reward mechanisms. This is the site of action of highly addictive drugs such as cocaine, nicotine and amphetamine. The work has high relevance for drug addiction, as understanding the function of the novel receptor signaling unit we have discovered will have significance for understanding the action of addictive drugs, and possibly identify mechanisms for treating the disorder.